Long-term effect of neoadjuvant radiotherapy in patients with locally advanced rectal mucinous adenocarcinoma: a population-based study of 1514 patients

Rectal mucinous adenocarcinoma (RMAC) is a rare and aggressive form of rectal cancer. The effectiveness of neoadjuvant radiotherapy (NRT) for RMAC has not been well studied, and the survival benefit remains controversial. The purpose of this work was to determine the prognostic role of NRT in patients with RMAC by propensity-score matching (PSM). A retrospective cohort study using the Surveillance, Epidemiology, and End Results from 2004 to 2015 was performed. In the multivariate analysis before PSM, NRT provided better OS (HR 0.61, 95% CI 0.52–0.71, p < 0.001) and CSS (HR 0.68, 95% CI 0.56–0.82, p < 0.001). Multivariate analysis after PSM (n = 844) confirmed that patients receiving NRT survived longer than those without NRT (OS: HR 0.60, 95% CI 0.50–0.78, p < 0.001 and CSS: HR 0.68, 95% CI 0.54–0.84, p < 0.001). Subgroup analysis indicated that NRT had significantly improved OS and CSS in stage II RMAC and OS in stage III RMAC after adjusting for various confounding factors.

nosed between 2004 and 2015 to explore in more detail the correlation between NRT and the long-term survival benefit of RMAC patients. The inclusion criteria were as follows: (1) patients diagnosed from 2004 to 2015; (2) histologically confirmed RMAC; (3) patients at stage II or III (pathological stage); (4) patients received preoperative radiotherapy followed by radical surgery or radical surgery alone; (5) age at diagnosis over 18 years. Based on whether patients received radiotherapy or not before radical surgery, the entire cohort was further divided into neoadjuvant radiotherapy (NRT) and non-neoadjuvant radiotherapy (non-NRT) groups. Finally, 1514 cases were included in our study (Fig. 1).
Outcome definition. The primary endpoints were overall survival (OS) and cancer-specific survival (CSS).
OS was defined as the time from diagnosis to death from any cause. CSS was defined as the time from the date of diagnosis to the date of death from rectal cancer or the latest follow-up.
Propensity score matching. Propensity score matching (PSM) is a reliable statistical method for reducing selection bias in observational studies and achieving the balance of covariates across the study groups 16 . Baseline characteristics between the NRT group and the non-NRT group were matched using nearest neighbor matching (1:1) with a caliper of 0.2.

Statistical analyses.
The chi-square test was used to compare the patients' baseline characteristics between the groups. Cox proportional hazard models were used to determine the prognostic factors for OS and CSS. Kaplan-Meier survival analysis and log-rank tests were performed to compare the survival among different groups. All tests were two-sided, and P-value < 0.05 was applied to indicate statistical significance. All statistical analyses and graphics were performed using Statistical Product and Service Solutions (SPSS) software (ver.26.0), R software (ver.3.6.3) and GraphPad Prism software (ver.8.0).  www.nature.com/scientificreports/ (81.4% and 85.4% respectively). For patients aged ≤ 60 years, they were more likely to receive NRT compared with patients aged > 60 years (83.1 vs. 58.7%). The proportion of patients with stage III RMAC receiving NRT is higher than those with stage II RMAC (73.2% vs. 61.1%). To balance the distribution of baseline characteristics, PSM was used. After matching, there was no significant differences between case and control groups except for chemotherapy (Table 1).
Survival analyses in the whole SEER cohort. OS and CSS for the entire cohort were 57.9% and 65.2% at 5 years, respectively, and 41.7% and 55.0% at 10 years, respectively. All baseline characteristics were selected for univariable and multivariate analysis to assess the effect on OS and CSS. Univariate analysis showed that older age (≥ 60 years), lager tumor size (> 5 cm), higher T stage (T4), N stage (N2), and number of LND < 12 were associated with worse OS and CSS, while NRT and chemotherapy were strongly associated with better survival ( Table 2). In multivariate analysis, NRT was an independent prognostic factor for OS and CSS (OS: HR 0.61, 95% CI 0.52-0.71, p < 0.001; CSS: HR 0.68, 95% CI 0.56-0.82, p < 0.001) ( Table 2). Furthermore, Kaplan-Meier curve analysis revealed that patients who received NRT had better OS (P < 0.001) and CSS (P = 0.001) than those who did not ( Fig. 2A, B).
Survival analysis in propensity score-matched cohort. In the matched cohort, univariate analysis showed similar prognostic factors for OS and CSS to the unmatched cohort: age, tumor size, T stage, N stage, number of LND, NRT and chemotherapy ( Table 3). The multivariate analysis showed that NRT was an independent prognostic factor for OS and CSS; OS (HR 0.60, 95% CI 0.50-0.78, p < 0.001) and CSS (HR 0.68, 95% CI 0.54-0.84, p < 0.001) of NRT-receiving patients were better than their non-NRT-receiving counterparts (

Subgroup analysis according to TNM stage.
To further identify patients who may benefit from NRT, subgroup analyses according to TNM stage were performed. Of note, no significant difference was found between the NRT and non-NRT groups in terms of baseline characteristics except for chemotherapy after PSM (all p > 0.05, Table 4). Per Kaplan-Meier survival analysis, NRT significantly improved OS and CSS of stage II RMAC compared to their non-NRT-receiving counterparts (OS: p < 0.001, CSS: p = 0.010). In addition, NRT improved OS, but not CSS, in stage III patients (OS: p = 0.014, CSS: p = 0.203) (Fig. 3).

Discussion
During the first decade of the twenty-first century, preoperative radiotherapy became the standard neoadjuvant strategy for LARC due to its advantage on increasing sphincter preservation rate and reducing local recurrence [17][18][19] . Growing evidence has demonstrated that mucinous adenocarcinoma is a distinct histologic subtype with different natural history, biological behavior, pathologic and molecular features, which may make them respond differently to treatment compared to their non-mucinous counterparts [20][21][22][23][24] . Moreover, RMAC has a higher ratio of lymph node infiltration and peritoneal implant 25,26 . However, given the rarity of RMAC, there are few studies about treatment for patients with RMAC and, to date, RMAC is treated with the principles that are developed for more common non-mucinous rectal cancer. In the era of precise medicine and personalized treatment, it is extremely important to understand the prognostic value of NRT for RMAC. To our knowledge, this is the first population-based study using PSM analysis to assess the role of NRT in locally advanced RMAC. Most studies showed that the histology of mucinous adenocarcinoma in rectal cancer served as a biomarker for poor prognosis after preoperative chemoradiotherapy. Sengul et al. demonstrated that patients with RMAC had obvious tumor regression and a decreased transrectal ultrasound score after receiving preoperative irradiation and 5-FU infusion, although not to the same degree as in non-mucinous adenocarcinoma 15 . Simha et al. came to a similar conclusion where RMAC patients were treated with preoperative radiation and 5-FU plus leucovorin chemotherapy 27 . In addition, Hugen et al. showed that short-term preoperative radiotherapy could narrow the survival gap between mucinous and non-mucinous rectal adenocarcinoma and led to a decrease in local recurrence of RMAC 28 . However, none of these studies had evaluated the efficacy of NRT on long-term outcomes in patients with RMAC or performed subgroup analyses to further investigate the relationship between NRT and survival outcomes. The results of the present study showed that NRT was independently associated with better OS and CSS, both before and after PSM for the entire cohort. Furthermore, the subgroup analysis revealed that NRT had significantly improved OS and CSS in stage II RMAC and OS in stage III RMAC. The reason why NRT did not result in better CSS in stage III RMAC might be attributed to the inadequacy of cases; another possible explanation was that NRT was less likely to influence the CSS of this subgroup, who tended to be more advanced at baseline and had worse prognosis.
Our study used a population-based cancer registry; unlike single-institution studies, which inevitably have a referral bias, the SEER database provides a more realistic clinical practice environment with information from all levels of healthcare institutions. Although there are many strengths of this study including the large sample size, PSM test and subgroup analysis, we acknowledge some limitations to our study. First, there were no information regarding preoperative radiotherapy in the SEER database, including clinical target volume and radiation regimen, which may cause confusion. Second, data on chemotherapy, such as regimen and courses, were also unavailable, so that further case-control studies failed to be performed. Finally, the SEER database did not include local recurrence and disease-free survival, which made the local control benefit of radiation therapy www.nature.com/scientificreports/ unanalyzable. However, the association of NRT with better OS and CSS in stage II RMAC and better OS in stage III RMAC was sufficient to support the advantage of preoperative radiotherapy.

Conclusions
In conclusion, our results have shown that locally advanced RMAC can gain survival benefit from NRT, which could provide better OS and CSS in stage II and better OS in stage III RMAC. Nonetheless, our results should be interpreted with caution, and further prospective clinical trials are needed, given the observational bias caused by their retrospective nature. www.nature.com/scientificreports/

Data availability
The data that support the findings of this study are available from Surveillance, Epidemiology, and End Results (SEER) database (https:// seer. cancer. gov/) but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the corresponding author upon reasonable request and with permission of SEER database.